When the cornea is damaged due to severe trauma or disease, its normally smooth contour and optical clarity are often compromised, resulting in loss of vision. The standard of care in these cases involves corneal transplantation; however, donor tissue remains limited in most parts of the world and unavailable to many who need it. Although there have been promising results in the creation of biosynthetic tissue graft materials, recapitulating the long-term transparency, biomechanics, and regenerative capacity of a human donor cornea remains a formidable challenge. Previous research has demonstrated that stem cell therapy could be an alternative option to donor tissue, and it was shown that transplanted corneal stromal stem cells (CSSCs) can prevent corneal scar formation and restore corneal transparency. However, cell viability following simple injection remains low due to mechanical damage during the injection procedure. In addition, CSSC phenotype post-transplantation and the mechanism behind their regenerative potential remain unknown. Therefore, new methods for delivering CSSCs and understanding how they respond to extracellular environment to facilitate corneal transparency are needed. In this proposed research, I will develop a bioothogonally crosslinked collagen gel with tunable mechanical properties that I hypothesize can successfully deliver CSSCs to the wounded cornea, stabilize the wound, and promote rapid re-epithelization while maintaining corneal transparency. We have previously demonstrated that bioorthogonal crosslinking can improve the mechanical stability of collagen while avoiding the potential off-target and cytotoxic effects of typical crosslinking chemistries. Here, I will determine how the culturing CSSCs in the gel matrix affects the gel?s mechanical properties and prolonged transparency over time as a function of bioorthogonal crosslinking density. I will also characterize how the crosslinking density affects the phenotypic transition of CSSCs to keratocytes and determine how the 3D culture conditions affect the CSSCs transcriptome. Then I will evaluate the paracrine role of the CSSCs in re-epithelization of the damaged cornea, including differences in the expression of growth factors and how the engineered gel is integrated into native tissue. This will advance our basic understanding of how CSSCs contribute to corneal transparency by remodeling their extracellular matrix and how their surrounding microenvironment influences their differentiation and regenerative potential. Ultimately, this work could provide a platform technology to enable wound healing inside and outside the eye.